GSM6680887: scRNA-seq of patient 2 pre NMP; Homo sapiens; RNA-Seq
Liver biopsies taken pre (T0) and at the end (T1) of NMP as well as after reperfusion (T2) (n=8 livers) were immediately minced into small pieces (<1 mm) on ice and subsequently digested enzymatically for 10 minutes at 37°C with agitation using the BD TuDoR™ dissociation reagent (BD Biosciences). The single-cell suspension was filtered through a 100 µM cell strainer and red blood cells were removed with the BD Pharm Lyse™ (BD Biosciences) lysing solution according to the manufacturer´s protocol. Cell viability was measured with the BD Rhapsody™ scRNASeq platform (BD Biosciences) using Calcein-AM (Thermo Fisher Scientific) and Draq7 (BD Biosciences). The freshly isolated single-cell suspension was immediately processed (<30 minutes from tissue dissociation to cell lysis) and whole transcriptome amplification (WTA) sequencing libraries were generated according to the BD Rhapsody™ single-cell WTA protocol. The quality of the obtained sequencing libraries was verified with the 4200 TapeStation system (Agilent) and the Qubit dsDNA HS (High Sensitivity) assay kit (Thermo Fisher Scientific). Sequencing was performed on the NovaSeq 6000 System platform (Illumina) with the S1 Reagent Kit v1.5 (200 cycles, 68 bp index read 1; Illumina) at a calculated sequencing depth of 50.000 reads/cell.